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Could a new twist to anti-amyloid immunotherapy—one that slowly drips the antibodies directly into the ventricles of the brain—solve some of the controversial issues in passive immunization? That is the implication of new work by Lisa Shafer and colleagues at the medical device company Medtronic in Minneapolis, Minnesota. The scientists report that prolonged infusion of low-dose anti-Aβ antibodies into the intracerebroventricular space of aged AD transgenic mice cleared established plaques and improved performance in behavior tests while at the same time avoiding accumulation of amyloid in blood vessel walls and associated microhemorrhage. These are two unwanted side effects that have been reported to be associated with passive immunization through peripheral routes. The work appears in this week’s PNAS online edition.

With passive immunization, mobilization of parenchymal plaques by Aβ antibodies has in some studies increased vascular Aβ deposition and led to the appearance of microbleeds (see ARF news story on Pfiefer et al., 2002; Wilcock et al., 2004; Racke et al., 2005). More recently, a study suggested that at least part of the vascular complication may stem from giving high concentrations of antibody, since lower doses given over six months were found to clear plaque and vascular amyloid without triggering bleeding in mice (see ARF related news story).

In the new study, lead author Deepak Thakker and colleagues looked to see what would happen if they bypassed the circulation altogether and delivered antibodies directly into the intracerebroventricular (ICV) space. They compared the effects of the 6E10 antibody, which binds the N-terminus of Aβ, in aged Tg2576 mice, administered for five weeks either by an implanted osmotic mini-pump into the ventricular space, or by repeated intraperitoneal (IP) injections. (Medtronic markets numerous pumps for peripheral and CNS applications.) At the end of the study, both groups of mice showed similar and extensive clearance of amyloid plaque from the cortex and hippocampus, and a reduction in astrocyte clusters and dystrophic neurites around plaques. These histological changes were accompanied by improvement to the levels of normal mice in the cued fear-conditioning test. The results were striking because the central dosing regimen used 10 times less antibody than the injection route (0.2 mg total dose vs. 2 mg for IP injections).

In contrast to their similar effects on plaques, the two regimens had a different impact on vascular amyloid. Tg2576 mice develop age-dependent CAA (as do many people with AD). This vascular pathology grew by more than half in response to systemic antibody treatment, coupled with a doubling of the frequency of cerebral microhemorrhages. The ICV antibodies, on the other hand, reduced CAA by 40-45 percent, and did not increase the number of hemorrhages.

With both treatments, antibodies infiltrated the brain tissue and similarly activated microglia around plaques. This suggests that the difference in vascular amyloid was not due to microglia-dependent mechanisms, the authors write. What did differ was peripheral clearance. Antibody levels and plasma Aβ were both significantly elevated by systemic antibody treatment, but not by ICV infusion. The results suggest that antibody-mediated clearance of brain Aβ through blood vessels may exacerbate CAA and associated microhemorrhages. This idea jibes with a recent study showing that people who received a now-discontinued Elan Aβ vaccine showed a transient worsening of CAA (Boche et al., 2008). Current human immunotherapy trials monitor patients closely for signs of microhemorrhage.

The lack of evidence for peripheral clearance after ICV antibody administration suggests that this treatment does not engage the “peripheral sink” clearance mechanism. Instead, the authors speculate, antibodies in the CSF might represent a “CSF sink,” and clear Aβ via a pathway that bypasses the cerebral vasculature. The observed slower rate of clearance could be another reason why low intracerebroventricular doses did not increase CAA, the authors hypothesize. They bolstered this idea by showing that a single high dose of antibody (12 micrograms) given via the ICV route rapidly cleared plaques, but also caused a transient increase in CAA and microhemorrhages.

The net result of ICV delivery is to increase the safety profile of passive immunization at later stages of disease, the authors said. “Preliminary data from recent Phase 1/2 Elan/Wyeth trials, along with recent preclinical work in AD mouse models, suggest that immunotherapy may be more effective if initiated in early stages of the disease, when the amyloid accumulation is less extensive and the cerebral vasculature is not highly compromised,” Thakker told ARF. “With the dosing paradigm and delivery strategy described in the paper, we are able to see a significant decrease in the vascular amyloid pathology, parenchymal amyloid pathology, and associated neuropathology, and a reversal of the behavioral deficits at a later disease stage in the transgenic mouse model.”

From a safety perspective, Shafer told ARF, “Preclinical and clinical work with systemic administration of anti-Aβ antibodies indicates an increased risk of accumulating amyloid in the vasculature and development of microhemorrhages. Our data replicates the increase in vascular amyloid and associated microhemorrhages with systemic administration of antibodies in the mouse model, and extends to show that these undesirable pathological indices are actually decreased with ICV delivery of antibodies.”

As for the feasibility of translating this delivery method to humans, the technology is in early clinical testing. NeuroNova of Sweden and Medtronic have started a Phase 1 trial using a programmable infusion pump linked to an ICV catheter to deliver vascular endothelial growth factor for the treatment of ALS.

This same system could be applicable to an Alzheimer disease indication, Shafer told ARF. In addition, she noted that surgical placement of an ICV shunt is a common procedure in elderly patients to treat normal pressure hydrocephalus, which causes a form of dementia that reverses when excess CSF is drained. Medtronic markets such a shunt. “There you are pulling from the ventricular space and here you’d be pushing in, but as far as a neurosurgical approach, the shunt provides some precedent,” Shafer said. Other routes of entry to the CSF are also possible. For that, Medtronic already has an FDA-approved device for chronic intrathecal infusion of drugs for the treatment of pain and spasticity. Furthermore, if delivery to the CSF does not work, Shafer says, infusion directly into brain tissue could be a possibility, as was done in a trial of the neurotrophic factor GDNF for Parkinson disease (see ARF related news story).

But, Shafer cautioned, the research is at an early stage. “We are encouraged by the results and especially the safety profile in transgenic mice, but we don’t know how that will translate. We are still determining the next step.” She declined to say if her group has tested any other antibodies or other models, or whether Medtronic plans to collaborate with other companies to deliver existing antibodies to humans.—Pat McCaffrey

Comments

This is a very important paper that was performed very professionally and addresses a critically important question regarding immunotherapy for Alzheimer disease. The observation that centrally administered antibodies have different characteristics than peripherally administered antibodies is a critical point, first identified by Vasilevko et al. (2007). This manuscript very clearly and convincingly demonstrates that the ICV route effectively clears Aβ deposits, yet does not produce increased CAA or hemorrhage, while the same antibody administered systemically produces comparable clearance, yet does increase CAA and hemorrhage. This would imply that central administration may be a safer method for testing anti-amyloid immunotherapy as a treatment for Alzheimer disease.

An overwhelming number of studies have published results that do not increase CAA or hemorrhage, but rarely include the positive contrast to show the mice used are of appropriate age and genotype to exhibit such an effect if one were present with the treatment. The authors are to be commended for going to the effort and doing this study correctly (i.e., in aged mice). For what it’s worth, we have analyzed our own past studies using intracranial administrations and never observed buildup of vascular Aβ or hemorrhage (never published). The data here indicate that only very high exposures to centrally administered antibody results in vascular accumulation. Importantly, this vascular accumulation and microhemorrhage were found in the human brain with vaccines (Boche et al., 2008).

There is an intriguing mismatch between the systemic and central doses used for Aβ clearance. The argument made by almost everyone is that 0.1 percent of plasma antibody enters brain. If true, the effective central dose of 6E10 when administered systemically was 0.002 mg. Yet the systemic administration provided dosing slightly more effective than 0.04 mg injected centrally. One potential explanation for therapeutic equivalence at different dosages could be clearance rates. Conceivably, the centrally administered antibody is cleared more rapidly due to microglial phagocytosis, effectively reducing the concentration of antibody.

The authors claim that the antibody-Aβ complex does not interfere with their ELISA, but since no dissociation steps are undertaken, this is unlikely to be the case. They claim to have 60 nM Aβ and the antibody concentration is roughly 200 nM. Thus, about 30 percent of antibody is occupied by Aβ. Our work finds that at these ratios there is increased Aβ and increased antibody concentration found when a simple acid dissociation step is employed prior to assay (Li et al., 2007). Ultimately, this is irrelevant to the arguments being made by the authors, but I think they need to prove that there is no interference if they are going to make the statement. In fact, I suspect the Aβ ELISA uses 6E10 as the capture antibody. If 6E10-Aβ plasma complexes enter the assay, they will not be detected due to masking.

In summary, a great paper with very convincing data. It would seem to support the use of central administration of antibodies as a test of the amyloid hypothesis of Alzheimer disease.

It is well known that at first anti-Aβ vaccination (active and passive) in mouse models of AD did not show clear side effects and subsequently several companies initiated active and passive vaccination trials in AD patients. There was only one unconfirmed report about lymphocyte infiltration detected in the brains of wild-type mice immunized with Aβ42 formulated in CFA/IFA followed by injection with pertussis toxin. Later, microhemorrhages in the cerebral vasculature have been observed in different strains of very old (21-26-month-old) APP/Tg mice injected weekly with high doses of anti-Aβ monoclonal antibodies, and the sites of microhemorrhage have been colocalized with cerebral vascular Aβ deposits. Collectively, these adverse events emphasized the need for further refinement of vaccines for AD in order to eliminate, or at least attenuate, the potential adverse events initiated by infiltration of autoreactive T cells and peripheral macrophages, as well as inflammation-induced cerebral vascular microhemorrhages. Accordingly, Thakker et al. initiated studies funded by Medtronic, Inc., and demonstrated in AD mouse model that delivery of anti-Aβ antibodies into the brain by direct intracerebroventricular (icv) infusions is more effective than systemic delivery of the same 6E10 monoclonal antibody. In general, these data supported previous published studies in which some groups demonstrated that both intracranial and peripheral administration of anti-Aβ antibodies could clear AD-like pathology in APP/Tg mice. What is new in this paper is that authors suggested that icv-administration of anti-Aβ antibodies is safer because it is reducing CAA and associated microhemorrhages.

While these results are interesting, we should be cautious because data from active or passive Aβ immunotherapy conducted with AD patients indicated that despite evidence of amyloid plaque removal, there is no indication of significant improvement of dementia in these patients. On top of that, from both clinical trials and animal studies we know that antibody could clear/decrease Aβ deposition without significant changes of tau pathology, neuropil threads, synaptic dysfunction, and cerebral amyloid angiopathy (CAA) even in areas where amyloid plaques had been removed. In sum, results from both clinical trials and animal studies indicate that Aβ-immunotherapy should be initiated before pathological forms of amyloid accumulated into the brain, and in this scenario icv-infusion is not feasible. Except for the difficulty associated with the delivery of anti-Aβ antibody to the brains of AD patients by icv infusions, one should expect that the high concentration of this antibody (40-200 μg) may bind a complement system in the brain and induce activation of these molecules (for example, C1q and C3). Although complement is one of the most critical defense systems of organism, its activation could induce profound brain tissue damage in AD patients. Thus, another precaution should be applied for the strategy based on icv-infusions of antibodies into the brains.

This is certainly a very well performed and interesting study. The authors argue that the engagement of central mechanisms and long-term intracerebroventricular infusion of 6E10 at a low dosage leads to the favorable outcome with reduced parenchymal plaques, cerebral amyloid angiopathy (CAA) and few microhemorrhages. It would be interesting to also investigate the effects of prolonged peripheral infusion of the same antibody in dose-response experiments. This would help determine if both factors are essential for the outcome.

Intraventricular infusion is seemingly both a risky and a complicated strategy, but it is clearly feasible as shown with rituximab for the treatment of lymphoma (Pekls et al., 2003).

Careful design of an antibody with respect to pharmacokinetics, CAA-binding and a well- adjusted dosage (Schroeter et al., 2008) might be alternative ways to reach the goal of a safe and efficacious immunotherapy for Alzheimer disease.